Adenoviruses (Ads) are a relatively well characterized, homogeneous group of viruses. Roughly 100 different adenoviruses, including nearly 50 serotypes isolated from humans, have been identified to date.
Most common serotypes of Ads are nonpathogenic, physically and genetically stable, can be grown to very high titres (concentrated stocks with 10.sup.11 -10.sup.12 PFU/ml of infectious virus are easy to obtain) and easily purified by isopycnic centrifugation in CsCl gradients. The Ad genome is readily manipulated by recombinant DNA techniques, and the proteins encoded by foreign DNA inserts that are expressed in mammalian cells will usually be appropriately glycosylated or phosphorylated, unlike recombinant proteins expressed in bacteria, yeast, and some insect cells. Although human Ads replicate most efficiently in human cells of epithelial origin, these viruses infect almost any mammalian cell and express at least some viral genes. Unlike retroviruses, Ads will infect, and are expressed in, nonreplicating cells. Thus, Ad-based vectors may be useful for gene delivery, expression, and gene therapy.
Ad vectors have been constructed by ligation or recombination of viral DNA with subgenomic viral sequences contained in bacterial plasmids. Berkner, K. L. & Sharp, P. A. (1983) Nucleic Acids Res. 11: 6003-6020; Haj-Ahmad, Y & Graham, F. L. (1986) J. Virol. 57: 267-274; Stow, N. D. (1981) J. Virol. 37: 171-180. This approach has several drawbacks, which include the time and technical difficultly required to produce viral DNA, the background of infectious parental virus which makes screening more labor intensive and, in the case of direct ligation, the limited availability of useful restriction sites due to the relatively large size of the adenovirus genome.
Another strategy has been to recombine two plasmids which together contain sequences comprising the entire Ad genome. A number of conditionally defective plasmid systems have been developed making the construction of vectors simpler and reducing the number of subsequent analyses required to identify recombinant viruses. McGrory, W. J., Bautista, D. S. & Graham, F. L. (1983) Virology 163: 614-617; Ghosh-Choudhury, G., Haj-Ahmad, Y., Brinkley, P., Rudy, J. & Graham, F. L. (1986) Gene 50: 161-171; Mittal, S. K., McDermott, M. R. Johnson, D. C., Prevec, L. & Graham, F. L. (1993) Virus Res. 28, 67-90.
The representative Adenovirus 5 ("Ad5") genome used in embodiments of the present invention is a 36 kb linear duplex. Its sequence has been published. (Chroboczek, J., Bieber, F., and Jacrot, B. (1992) The Sequence of the Genome of Adenovirus Type 5 and Its Comparison with the Genome of Adenovirus Type 2, Virology 186, 280-285; hereby incorporated by reference). The Ad5 genome contains a 100-150 base pair (bp) inverted terminal repeat ("ITR") at each end of the linearized genome. A terminal protein ("TP") of 55,000 daltons is covalently linked to the 5' end of each strand. Both the TP and the ITRs are thought to play a role in viral DNA replication. McGrory, W. J. et al. (1988), Virology, 163, 614-617 and Ghosh-Choudhury, G. et al. (1986), Gene, 50, 161-171 (hereby incorporated by reference). Ad5 has infected each human cell line tested, although some cells, such as lymphocytes, are relatively nonpermissive.
Four noncontiguous regions of the Ad5 genome are transcribed early in infection, prior to DNA replication. These regions are early region 1 (E1) (about 1.3-11.2 mu of or about position 198-4025 bp of a standardized genome, inclusive of the E1A enhancer region; Sussenbach, J. S., in Ginsburg (Ed.), The Adenoviruses pp. 35-124, 1984, Plenum Press) which is further divided into subregions E1A and E1B; early region 2 (E2), which encodes the DNA replicative functions of the virus; early region 3 (E3) (about 75.9-86.0 mU, or about 27,275-30,904 bp; Cladaras, C. and Wold, W. S. M. (1985) Virol. 140, 28-43; and early region 4 (E4). E1A is involved in turning on the other early regions and in regulating a number of host cell functions. E1B and E4 are primarily involved in shutting off the host cell's protein synthesis. E3 regulates the host cell's immune response to virus infection. Some of these early genes function to "turn on" later-expressed genes that are needed to replicate the genome and form viable viral particles.
The Ad virion has the ability to package up to 105-106% of the wild type genome length. Bett, A. J., Prevec, L., & Graham, F. L. (1993) Packaging Capacity and Stability of Human Adenovirus Type 5 Vectors, J. Virol. 67: 5911-5921. Larger genomes (e.g., 108% of the wild type in size), result in instability of the virus and poor growth rates. Id. This packaging ability allows the insertion of only approximately 1.8-2.0 kb of excess DNA into the Ad genome.
To package larger inserts, it is necessary to first delete portions of the viral genome. Parts of region E1 can be deleted, and the resulting viruses can be propagated in human 293 cells. (293 cells contain and express E1, complementing viral mutants that are defective in E1.) Foreign nucleic acids can be inserted in place of E1, in Ad5 genomes that contain E1 deletions of up to 2.9 kb, to yield conditional helper-independent vectors with a capacity for inserts of 4.7-4.9 kb.
Viruses with a region E3 deletion can also replicate in cultured human cells such as HeLa or KB and infect and be expressed in animals including humans. A deletion of a 3.0 kb E3 sequence has been reported, without a concomitant insertion. Ranheim, T. S., Shisler, J., Horton, T. M., Wold, L. J. Gooding, L. R., and Wold, W. S. M. (1993) J. Virol. 67, 2159-2167.
Among the methods developed to date there is no simple procedure for generating vectors that utilize both E1 and E3 deletions. In addition, the vectors that do utilize either E1 or E3 deletions can accomodate only relatively small inserts. To simplify the production and use of Ad vectors that can tolerate larger fragments, we have developed a new methodology based on a series of bacterial plasmids that contain most of an Ad viral genome.